Table 3.
Characterization technology of bioprinted constructs. Value of 3D bioprinting for cancer modelling. + for pros and − for cons.
| Methods | Description | Pros and Cons | Markers | REF | |
|---|---|---|---|---|---|
| Microscopy | |||||
| Light | Phase contrast | Monitoring of proliferation and morphology of cells | +: • Nondestructive • No markers are added • Low cost • Easy with transparent gels (GelMA, matrigel) −: • No possibility to identify subcellular structures • Difficult with opaque or non-transparent gels (e.g.,: alginate with nanocellulose) |
Not suitable | [100,101,102] |
| Bright field | The transmission of light is more or less attenuated depending on the density or marking of the sample | +: • Suitable for large samples −: • Requires histological staining • Preparation of sample • Quantification of thick sample |
Hematoxylin–eosin Masson’s trichrome Trypan blue |
[101,102,103] | |
| Fluorescence | LSM Epifluorescence Confocal |
The use of a fluorescent marker is necessary to highlight a subcellular structure; possibility of monitoring structures over time (if vital markers) | +: • Monitoring of many possible structures −: • Requires cutting for oversized constructions for epi and confocal microscopy • Need to fix for certain markers • Important autofluorescence for chitosan or alginate/cellulose hydrogels in UV |
Live/dead staining Or calcein AM/propidium iodide Or ethidium homodimer Active-caspase3/7 green Hoechst 33342 HIF1-α, Ki67 |
[108,109,110,111,144] |
| Electronic | Scanning | Surface is scanned with a beam of electrons, emitted signal provides images | +: • High resolution −: • The preparation procedure is tedious • Frequent preparation artifacts (collapse) |
Not suitable | [102] |
| Transmission | The part of beam of electrons is transmitted into specimens allowed to obtain images | Not suitable | [102,115] | ||
| Flow cytometry | |||||
| Flow cytometry | Analysis of physical parameters (size and granularity) for each cell but also the level of fluorescence | +: • Quantitative analysis −: • Disaggregation can be a problem • Necessity to have a large cell number due to loss of cells during dissociation |
7-AAD CFSE |
[102,139] | |
| Spectroscopy | |||||
| Spectrometry or fluorimetry | Production or utilization of a fluorescent or chromatic compound | +: • Well-described for 2D culture and frequently used • Can be used for kinetic monitoring −: • Ensure that the efficiency is adapted for 3D |
ACP, LDH, prestoblue, alamar blue, DNA content | [112,119,120,121] | |
| Molecular biology | |||||
| RTqPCR Western blot |
Quantification of gene expression at mRNA or protein level | +: • Quantitative analysis • Easier by using the enzymatic method on natural inks (e.g., collagenase for GelMA or ColMA, hyaluronidase for hyaluronic acid) −: •Adaptation of the homogenization and extraction protocol to obtain an adequate quantity and quality of RNA/proteins for analyses |
Bax/Bcl2 HIF1-α, Ki67 |
[103,115,118] | |
| Metabolism | |||||
| GC–MS (Gas chromatography–mass spectrometry) | Detection of molecules of interest according to their mass/charge ratio after ionization | +: • Considerably less cellular material compared to NMR, high sensitivity, −: • Use of radioisotopes, complex sample preparation, high cost |
13C-Glucose | [129,132] | |
| NMR (nuclear magnetic resonance) spectroscopy | Determination of the composition of a sample by applying a magnetic field via the orientation of the nuclear spins of the atoms | +: • High reproducibility, sample can be analyzed directly, low cost −: • Use of radioisotopes, low sensitivity |
[130,131] | ||
| PET scan (positron emission tomography) | Injection of a radiographic tracer and monitoring by imaging to detect localization of [18F]FDG | +: • Classically used in medicine, monitoring over time −: • Low resolution (1.5 mm) |
[18F]FDG | [120,125] | |
| Seahorse | Quantification of the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) | +: • High sensitivity (from 5000 cells, theoretically), possibility to test many conditions in parallel −: • Difficulties in normalizing results, limited number of injections, limited sample thickness |
Not suitable | [126,128] | |
7-AAD: 7-ADDminoactinomycin; [18F]-FDG: 18F-2-Fluor-2-deoxy-D-glucose; ACP: acid phosphatase assay; CFSE: carboxyfluorescein succinimidyl ester; CTV: celltraceviolet; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; pNPP: p-nitrophenyl phosphate; PET: positron emission tomography; WST: water-soluble tetrazolium; XTT: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.